Biotechnology for Biofuels最新文献

Food loss and waste (FLW) generated by unsustainable linear food systems are major contributors to greenhouse gas (GHG) emissions. Although microalgal lipid production has advanced significantly for applications such as biofuels and high-value polyunsaturated fatty acids (PUFAs), the use of FLW as an alternative feedstock to cultivate lipid-rich microalgal biomass within a circular bioeconomy remains insufficiently explored. This review critically evaluates the feasibility of converting FLW into nutrient-rich media for microalgae cultivation, with particular focus on its effects on biomass productivity and lipid accumulation. Pre-treatment methods for food waste are essential to enhance nutrient recovery, especially of carbon sources, and significantly influence subsequent microalgae cultivation. These methods affect the bioavailability of key nutrients, particularly the carbon-to-nitrogen-to-phosphorus (C/N/P) ratio, which regulates metabolic pathways involved in lipid biosynthesis. Despite encouraging laboratory-scale outcomes, large-scale implementation remains constrained by feedstock heterogeneity, high energy demands during harvesting and lipid extraction, and regulatory challenges. To overcome these barriers and facilitate scale-up, this review highlights integrative strategies such as metabolic engineering, automated cultivation systems, and a closed-loop microalgae-based biorefinery. Moreover, life cycle assessment (LCA) is emphasized as a tool to assess environmental performance and inform policy decisions, supporting alignment with Sustainable Development Goals (SDG 12 and SDG 13).

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Background

As outlined by the Intergovernmental Panel on Climate Change, we need to approach global net zero CO₂ emissions by approximately 2050 to prevent warming beyond 1.5 °C and the associated environmental tipping points. Future microbial electrosynthesis (MES) systems could decrease net CO₂ emissions by capturing it from industrial sources. MES is a process where electroactive microorganisms convert the carbon from CO₂ and reduction power from a cathode into reduced organic compounds. However, no MES system has attained an efficiency compatible with a financially feasible scale-up. To improve MES efficiency, we need to consider the energetic constraints of extracellular electron uptake (EEU) from an electrode to cytoplasmic electron carriers like NAD⁺. In many microbes, EEU to the cytoplasm must pass through the respiratory quinone pool (Q-pool). However, electron transfer from the Q-pool to cytoplasmic NAD⁺ is thermodynamically unfavorable. Here, we model the thermodynamic barrier for Q-pool dependent EEU using the well-characterized bidirectional electron transfer pathway of Shewanella oneidensis, which has NADH dehydrogenases that are energetically coupled to proton-motive force (PMF), sodium-motive force (SMF), or uncoupled. We also tested our hypothesis that Q-pool dependent EEU to NAD⁺ is ion-motive force (IMF)-limited in S. oneidensis expressing butanediol dehydrogenase (Bdh), a heterologous NADH-dependent enzyme. We assessed membrane potential changes in S. oneidensis + Bdh on a cathode at the single-cell level pre to post injection with acetoin, the substrate of Bdh.

Results

We modeled the Gibbs free energy change for electron transfer from respiratory quinones to NADH under conditions reflecting changes in membrane potential, pH, reactant to product ratio, and energetically coupled IMF. Of the 40 conditions modeled for each method of energetic coupling (PMF, SMF, and uncoupled), none were thermodynamically favorable without PMF or SMF. We also found that membrane potential decreased upon initiation of EEU to NAD⁺ for S. oneidensis on a cathode.

Conclusions

Our results suggest that Q-pool-dependent EEU is both IMF-dependent and is IMF-limited in a proof-of-concept system. Because microbes that rely on Q-pool-dependent EEU are among the most genetically tractable and metabolically flexible options for MES systems, it is important that we account for this thermodynamic bottleneck in future MES platform designs.

Advancing biomethane production from anaerobic digestion (AD) is essential for building a more reliable and resilient bioenergy system. However, incomplete conversion of lignocellulose-rich agricultural waste remains a key limitation, often leaving energy-dense residues in the digestate by-product. In this study, we introduce a novel application of chelator-mediated Fenton (CMF) post-treatment to recover untapped biomethane potential from these recalcitrant residues, representing a significant departure from conventional pre-treatment strategies. By systematically varying pH, iron-chelator concentration, and hydrogen peroxide dosage, we identified reaction conditions (pH 6–8, 5 mM Fe²⁺-dihydroxybenzene, 3–4 wt.% H₂O₂) that enhanced lignocellulose deconstruction and increased dissolved organic carbon (DOC) availability for methanogenesis. CMF post-treatment led to up to a tenfold increase in biomethane potential compared to untreated controls. Microbial community analysis revealed enrichment of cellulolytic species, suggesting enhanced hydrolytic activity as a driver of improved conversion. Application of the CMF post-treatment method to isolated poplar lignin further demonstrated its versatility for diverse lignocellulosic substrates. These findings position CMF post-treatment as a promising strategy to enhance AD efficiency and valorize digestate.

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Rhodotorula glutinis is an important oleaginous yeast that can synthesize various valuable compounds, including carotenoids, lipids, and exopolysaccharides. The effect of combined heat stress and glucose starvation on carotenoid biosynthesis in R. glutinis was investigated in this study. Carotenoid production in R. glutinis was promoted by heat stress, and this effect was further enhanced when glucose starvation was applied to the strain. The results of multiomics analysis revealed that the effects of heat stress and glucose starvation on promoting carotenoid biosynthesis appeared to be additive, with the combined stress leading to a further increase in reactive oxygen species (ROS) levels and a reduction in enzymatic antioxidant capacity, while carotenoid biosynthesis was prioritized simultaneously. The key responses of R. glutinis to combined stress include the regulation of the cell cycle and energy metabolism, maintenance of membrane integrity, an increase in ROS scavenging capacity, and non-enzymatic antioxidant activity. Additionally, several candidate genes and metabolites associated with the combined stress response were identified. To summarize, we provided new insights into optimizing fermentation processes for increased carotenoid production in Rhodotorula glutinis and established a molecular basis for further genetic engineering to increase carotenoid yield.

Background

Glycoside Hydrolase family 2 (GH2) is one of the largest and most functionally diverse carbohydrate-active enzyme families. This functional diversity is an obstacle to accurate functional prediction by family assignment and has led to the accumulation of erroneous annotations in non-curated databases.

Results

We explored the sequence space of the GH2 family using Sequence-Similarity Networks coupled with closeness centrality to identify 23 subfamilies. The analysis suggests that the GH2 family evolved via multiple duplications followed by neofunctionalization events, with two main activities, β-glucuronidase and β-galacturonidase, re-emerging from likely flexible/reversible ancestors, while an early diverging branch gave birth to several subfamilies with unique activities. To increase the predictive power of subfamily assignments, we biochemically characterized seven members of four of the five subfamilies without previously reported activity.

Conclusions

The GH2 subfamilies showing high functional homogeneity will enable more precise functional predictions, while our work highlights subfamilies that require further biochemical and structural investigations.

To stay within the planetary boundaries circularizing economy by utilizing residues is key. Bioprocesses can use abundant, but complex biogenic residues, giving access to various value-added products. To advance circularization, the feasibility of exploiting diverse biogenic residues as feedstocks for different, yet specific, bioprocesses needs to be assessed. Exemplifying the national level in Germany, we categorized biogenic residues compiled in the DE Biomass Monitor regarding their composition and feedstock potential in a resource matrix, detailing their constituents and the quality of available data. Three biotechnological processes, making use of lignin, non-fibrous carbohydrates, and oil, respectively, served as model processes to assess the biogenic production potential. By developing material flows based on state-of-the-art conversion routes, we found that residue-based production via all three example processes could meet national demands of specific polymer bricks, medium chain carboxylates, and platform chemicals, respectively, when mobilizing only 20–30% of possible raw materials. The accruing side streams underline the importance of cluster approaches early in bioprocess development. Specific challenges for fully exploiting the potential of biogenic residues were identified, including legal and acceptance issues, the need for considered biomass decomposition in interweaved production lines, and residue availability and management. This study provides an example-based framework for integrating biogenic residues with biotechnological production, using the resource matrix and an initial material-to-product estimation to advance a circular bioeconomy.

Background

Overcoming lignocellulose recalcitrance to enzymatic or chemical processing is a prerequisite for biorefinery applications. Expansins and loosenins are non-lytic proteins that could assist reducing this recalcitrance by disrupting the intermolecular contacts between plant cell wall components. Here, immunolocalization with fluorescence and transmission electron microscopy (TEM) were used to study the ability of a Bacillus subtilis expansin-like protein (BsEXLX1), a Phanerochaete carnosa loosenin protein (PcaLOOL12) and a fusion protein of PcaLOOL12 with the carbohydrate-binding module 63 (CBM63) of BsEXLX1 (i.e., PcaLOOL12-CBM63) to bind secondary cell walls (SCW) of aspen fibres, including fresh aspen wood, milled wood fibres (MWF) and MWF subjected to subcritical water extraction.

Results

The immunofluorescence labelling of fresh wood samples showed a weak signal for PcaLOOL12 and a strong signal for BsEXLX1 and PcaLOOL12-CBM63, suggesting the importance of CBM63 for protein adsorption to SCW components. TEM analysis after immunogold labelling revealed the presence of BsEXLX1 and PcaLOOL12-CBM63 in all secondary cell wall layers. Pretreatment of wood samples with the proteins reduced the binding of glucomannan- and glucuronoxylan-specific monoclonal antibodies. Similarly, protein adsorption to MWF was higher before subcritical water extraction. Together, these results suggest the adsorption of BsEXLX1 and PcaLOOL12-CBM63 to SCWs was mediated at least in part by their interaction with hemicelluloses.

Conclusions

Our study demonstrates that microbial expansin-related proteins can bind to the secondary walls of aspen wood through potential interaction of CBM63 with hemicelluloses.

This study introduces a novel hybrid photobioreactor system that integrates an open raceway pond (ORWP) with a Nested-bottled photobioreactor (NB-PBR) in a closed-loop configuration to enhance microalgal biomass production and CO₂ fixation. The system facilitates continuous culture circulation, improving mass transfer and mixing efficiency while ensuring optimal light exposure and CO₂ dissolution. This design resulted in a 38% increase in dry mass (3.1 g/L) and improved mass transfer and mixing times by 16.6% and 15.3%, respectively. The optimized cultivation conditions led to a 39.9% enhancement in CO₂ fixation and an 8.7% increase in photosynthetic efficiency (Fv/Fm) compared to traditional systems. The strategic movement of poorly illuminated ORWP to the NB-PBR maximized light absorption and nutrient uptake, significantly boosting overall productivity. These findings highlight the potential of hybrid photobioreactor systems in improving microalgal growth efficiency and advancing sustainable algal cultivation for commercial applications.

Background

Many industrial applications of wood and woody biomass require harsh physicochemical pretreatments to improve the hydrophobicity and durability of the products. Environmentally friendly wood biorefineries necessitate the replacement of chemicals and energy-consuming wood processing. Here, our goal was to increase wood hydrophobicity via the ectopic expression of Jojoba (Simmondsia chinensis) wax ester synthase (ScWS) in poplar (Populus × canescens). We expressed ScWS under a wood-specific promoter (DX15), which naturally controls the expression of FASCICLIN-like ARABINOGALACTAN PROTEIN 15 (FLA15) in the xylem.

Results

In the DX15::ScWS lines, ScWS was highly expressed in wood but not in leaves. The transgenic lines exhibited normal photosynthesis and growth similar to the wild-type poplars. Compared with the wild-type poplars, the DX15::ScWS lines accumulated greater amounts of triacylglycerol in wood and a greater number of lipid droplets in ray parenchyma cells. The composition of the bark cuticle wax esters was unaffected. The wood of the DX15::ScWS lines showed greater water repellency and less swelling than that of the wild-type poplars. Furthermore, the DX15::ScWS lines had an increased expression of FLA15 and increased cell wall deposition in fibers, resulting in increased wood density.

Conclusions

Our results highlight the potential of combining the wood-specific DX15 promoter with ScWS to enhance the technological properties of poplar wood. Reduced wood hydrophilicity represents a significant improvement in wood quality. In addition, our results suggest that the overexpression of the DX15 promoter could be a promising strategy for improving lignocellulose biomass in plants. Since poplars are highly productive species that can be cultivated in short-rotation plantations, our results have high translational potential for advancing sustainable wood utilization for a wider range of applications.

Background

Itaconic acid is a valuable platform chemical with applications in polymer synthesis and other industrial sectors. Microbial fermentation offers a sustainable production route, involving two fungi such as Aspergillus terreus and Ustilago maydis. However, their employment in industrial bioprocesses for itaconic acid production is characterized by several challenges. Yarrowia lipolytica is a non-conventional yeast that shows suitability for industrial production and it is widely employed as heterologous host to obtain relevant metabolites. This study aimed to engineer Y. lipolytica for the selective production of itaconic acid by optimising intracellular metabolic fluxes and transport mechanisms.

Results

A metabolic engineering strategy was developed to prevent the secretion of citric and isocitric acids by blocking their transport at both mitochondrial and plasma membrane levels in Y. lipolytica strains. Specifically, the inactivation of YlYHM2 and YlCEX1 genes reduced secretion of citric and isocitric acid, enabling their accumulation in the mitochondria. Additionally, heterologous transporters from Aspergillus terreus (mttA and mfsA) and Ustilago maydis (mtt1 and itp1) were introduced to enhance the mitochondrial export of cis-aconitate and the extracellular secretion of itaconic acid. For the first time, complete gene set of the itaconate biosynthetic pathways from both fungal species were functionally expressed and compared in a yeast system with a similar genetic background. A synergistic increase in itaconic acid production was observed when both pathways were co-expressed, combined with the inactivation of native citric and isocitric transport. In contrast to previously engineered Y. lipolytica strains for itaconic acid production, the optimised strain obtained in this study does not require complex or nutrient-rich media, while achieving the highest product yield (0.343 mol IA/mol glucose) and productivity (0.256 g/L/h) reported in yeast, with minimal by-product formation.

Conclusions

By integrating transporter engineering and pathway diversification, this study demonstrates an effective strategy to enhance itaconic acid production in Y. lipolytica while minimising by-product formation. The findings provide new insights into organic acid transport in yeast and open avenues for further optimization of microbial cell factories for sustainable biochemical production.

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